Optical imaging in biomedical technology is widespread in preclinical drug development and becoming more necessary in preclinical disease models. Immunohistochemistry for research and clinical trials has long been performed with either alkaline phosphatase (AP) or horse-radish peroxidase (HRP), particularly in the areas of biomedicine and diagnostics. In some methods commonly used in both systems, a primary target-specific antibody binds to a tissue. Then, a secondary antibody conjugated with AP or HRP is added that binds to the primary antibody. Detection of the target is visualized by observing the reaction of the enzyme with a substrate that is added to the reaction mixture. In most cases, the enzyme acts on the substrate to produce a color change in the test sample due to precipitation of reaction products on the tissues. In most methods, detection is visualized with standard light microscopy techniques.
But, these systems have limitations. For example, the range of colors for distinguishing reaction products is limited, and distinguishing even two colors is sometimes problematic, while distinguishing greater than two is not currently feasible. Likewise, other than highly specialized systems, these methods are not used to generate fluorescent reaction products. Also, endogenous enzymes present in tissues (e.g., alkaline phosphatases) can lead to high backgrounds (i.e., “noise”). As a result, special blocking steps are required to prevent undesired reactions and/or prolonged staining procedures are required to kill endogenous enzymes prior to staining. Thus, improved methods for detecting tissue targets are needed.